The worldwide demand for high potency sweeteners is increasing and, with blending of different sweeteners becoming a standard practice, the demand for alternatives is expected to increase. The sweet herb of Paraguay, Stevia rebaudiana Bertoni, produces an alternative high potency sweetener with the added advantage that Stevia sweeteners are natural plant products. In addition, the sweet steviol glycosides have functional and sensory properties superior to those of many high potency sweeteners. These glycosides accumulate in Stevia leaves where they may comprise from 10 to 20% of the leaf dry weight. Stevioside and rebaudioside A are both heat and pH stable (Chang and Cook, 1983), and suitable for use in carbonated beverages and many other foods. Stevioside is between 110 and 270 times sweeter than sucrose, rebaudioside A between 150 and 320 times sweeter than sucrose
Early steps in steviol biosynthesis involve the plastid localized 1-deoxy-D-xylulose 5-phosphate (DXP) pathway, resulting in the formation of DXP from pyruvate and glyceraldehyde 3-phosphate by thiamine phosphate dependant DXP synthase (Totté et al. 2000), and leading ultimately to the synthesis of geranyl geranyl diphosphate (GGDP). Like all diterpenes, steviol is synthesized from GGDP, first by protonation-initiated cyclization to (−)-copalyl diphosphate (CDP) by CDP synthase (CPS) (Richman et al., 1999). Next, (−)-kaurene is produced from CDP by an ionization dependant cyclization catalysed by (−)-kaurene synthase (KS) (Richman et al., 1999). (−)-Kaurene is then oxidized at the C-19 position to (−)-kaurenoic acid, by a novel P450 mono-oxygenase. Steviol is produced by the hydroxylation of (−)-kaurenoic acid at the C-13 position, but the gene for this P450-dependant mono-oxygenase has not yet been isolated (Kim et al. 1996. Arch. Biochem. BioPhys. 332:223-230). Steviol glucosides are formed by four glycosylation reactions that start with steviol and end with rebaudoside A (Richman et al. 2005). The steps involve the addition of glucose to the C-13 hydroxyl, the transfer of glucose to the C-2′ and C-3′ of the 13-O-glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl.
There is a need to identify methods of producing cells having the capability to synthesize steviol from ent-kaurenoic acid for use in synthesis of sweet steviol glycosides. However, the gene encoding ent-kaurenoic acid 13-hydroxylase is not known. There is a need in the art for compositions and methods for producing ent-kaurenoic acid 13-hydroxylase. There is also a need in the art for compositions and methods of producing steviol and steviol glycosides. Further, there is a need in the art for compositions and methods for producing steviol and steviol glycosides in cells, in plants and in vitro.